The long-term goal of the proposed research is to understand the relationship between cell mechanics and barrier functions of the vascular endothelium. Changes in the permeability of endothelia are important in processes such as angiogenesis, arthrosclerosis and inflammation. The cytoskeleton of vascular endothelium is known to be highly dynamic and is thought to be the primary determinant of cellular mechanics. Further, changes in the cytoskeleton have been shown to directly affect the endothelial barrier. The hypothesis put forth here is that such cytoskeletal reorganization results in a remodeling of the local mechanical properties, which in turn will have important consequences for barrier function. Thus, we propose an effort to characterize the local mechanical architecture of the cortex of a model endothelial cell (Bovine Pulmonary Artery Cells), and study the mechanical remodeling of these cells. To achieve this goal we here propose a pilot project to develop the imaging methods necessary to visualize cortical mechanics and to establish a clear connection between the cytoskeleton and cortical mechano-architecture. This pilot project has two specific aims. First we will use atomic force microscopy to produce high-resolution images of the local mechanical architecture of the cortex in living endothelial cells. These images will reveal how the cortex of these cells is organized, and how it remodels with time. We will also characterize the local mechanics through indentation measurements, and relate these measurements to the cortical morphology. Second, we will determine the molecular components that determine the mechanical architecture of vascular endothelial cells by using biochemical labeling and pharmacological approaches. Correlated imaging between confocal microscopy of cells labeled with antibodies specific for actin, tubulin and other cytoskeletal components will be used to identify what cellular structures are responsible for the mechanical features seen in the AFM images. In addition, pharmacological agents will be used to disrupt specific structures, in particular cytoskeletal structures, and correlate that to loss of mechanical features. The successful completion of this project provides the necessary technology and scientific foundation for a complete study of cortical mechanics of the vascular endothelium, and mechanical remodeling in normal and diseased cells.